The human heart continuously undergoes slow cellular turnover through apoptosis of cardiomyocytes and proliferation of cardiac progenitor cells at a rate of approximately 1% turnover a year (see Bergmann, O., et al., Evidence for cardiomyocyte renewal in humans. Science, 2009. 324(5923): p. 98-102). This slow cardiomyocyte turnover is important for maintaining cardiac function from birth to old age; however, resident cardiac progenitor cells have shown little capacity for robust cardiac regeneration following myocardial injury such as that caused by a myocardial infarction (see Bolli, P. and H. W. Chaudhry, Molecular physiology of cardiac regeneration. Ann N Y Acad. Sci. 1211: p. 113-26). Thus, it is likely that any successful post-injury progenitor cell-based cardiac regeneration strategy would require administering progenitor cells such as stem cells from a source outside of the native injured cardiac tissue.
A major obstacle in developing cell-based regenerative strategies is the need to successfully transfer a sufficient number of therapeutic cells to the target location (Karp, J. M. and G. S. Leng Teo, Mesenchymal stem cell homing: the devil is in the details. Cell Stem Cell, 2009. 4(3): p. 206-16). For example, in the infarcted heart, transfer of therapeutic cells is challenged not only by the motion of the heart but also by its heightened electrical and structural instability (Fernandes, S., et al., Autologous myoblast transplantation after myocardial infarction increases the inducibility of ventricular arrhythmias. Cardiovasc Res, 2006. 69(2): p. 348-58). Despite these difficulties, some reports indicate that intramyocardial or intracoronary injection of potentially therapeutic cells following cardiac injury can result in modest improvement in cardiac function (see, e.g., Bolli, R., et al., Cardiac stem cells in patients with ischaemic cardiomyopathy (SCIPIO): initial results of a randomised phase 1 trial. Lancet, 2011. 378(9806): p. 1847-57; Price, M. J., et al., Intravenous mesenchymal stem cell therapy early after reperfused acute myocardial infarction improves left ventricular function and alters electrophysiologic properties. Int J Cardiol, 2006. 111(2): p. 231-9). However, to date, no study has reported the large scale engraftment of cells or functional cardiomyocyte regeneration in an infarcted heart.
An alternative to direct injection of potentially therapeutic cells is to place them in a biomatrix “patch” that can be affixed to a desired area of the heart, such as onto the epicardium. In this way, a patch pre-seeded with potentially therapeutic cells may release the cells, allowing them to migrate into the injured myocardium to facilitate regeneration directly, or may retain the cells in close proximity to the injury to facilitate regeneration indirectly (i.e., via paracrine factors) (see Gnecchi, M., et al., Paracrine mechanisms in adult stem cell signaling and therapy. Circ Res, 2008. 103(11): p. 1204-19). This approach has been attempted using patches made of synthetic materials (see Silva, E. A. and D. J. Mooney, Synthetic extracellular matrices for tissue engineering and regeneration. Curr Top Dev Biol, 2004. 64: p. 181-205), as well as naturally occurring biomaterials such as the extracellular matrix (ECM) remaining after decellularization of heart valves (Bader, A., et al., Tissue engineering of heart valves—human endothelial cell seeding of detergent acellularized porcine valves. Eur J Cardiothorac Surg, 1998. 14(3): p. 279-84), skeletal muscle (Borschel, G. H., R. G. Dennis, and W. M. Kuzon, Jr., Contractile skeletal muscle tissue-engineered on an acellular scaffold. Plast Reconstr Surg, 2004. 113(2): p. 595-602; discussion 603-4), or bovine dermis (Kouris, N. A., et al., Directed Fusion of Mesenchymal Stem Cells with Cardiomyocytes via VSV-G Facilitates Stem Cell Programming. Stem Cells Int, 2012. 2012: p. 414038). Notably, none of these patches are made from myocardial tissue, nor do any of them exhibit the unique structural characteristics of cardiac ECM.
The extracellular matrix (ECM) is the extracellular part of animal tissue that provides structural support to the cells, in addition to performing various other important functions. As such, it is the defining feature of connective tissue in animals. Fibroblasts play a central role in the synthesis and maintenance of the ECM. In vivo, the ECM has a 3-dimensional structure, which facilitates interaction on all sides of the cells that are associated with the ECM. Specifically, cells associated with the ECM may be in contact with ECM surfaces both above and below the cells. To accurately model ECM-cell interactions in vivo, any ECM structure that is used in vitro would likewise need to have a true 3-dimensional structure. A 3-dimensional ECM cannot be substantially flat, and would have a thickness of at least 20 μm.
The cardiac ECM is a unique 3-dimensional structure that facilitates the normal functioning of the heart. The arrangement of the cardiac ECM helps channel the contraction of each myocyte into one forceful contraction, ultimately ejecting blood from the ventricles into the circulation (see Akhyari, P., et al., Myocardial tissue engineering: the extracellular matrix. Eur J Cardiothorac Surg, 2008. 34(2): p. 229-41). Furthermore, the cardiac ECM has importance beyond providing structure to cardiac tissue. Specifically, it plays a role in cardiac wound healing (see Dobaczewski, M., et al., Extracellular matrix remodeling in canine and mouse myocardial infarcts. Cell Tissue Res, 2006. 324(3): p. 475-88; Jourdan-Lesaux, C., J. Zhang, and M. L. Lindsey, Extracellular matrix roles during cardiac repair. Life Sci. 87(13-14): p. 391-400), and may play a role in cardiac regeneration (see Akhyari, P., et al., Myocardial tissue engineering: the extracellular matrix. Eur J Cardiothorac Surg, 2008. 34(2): p. 229-41). Although the production of a thin (<0.1 μm), 2-dimensional putative cardiac ECM on the surface of culture plate has been previously reported (see VanWinkle, W. B., M. B. Snuggs, and L. M. Buja, Cardiogel: a biosynthetic extracellular matrix for cardiomyocyte culture. In Vitro Cell Dev Biol Anim, 1996. 32(8): p. 478-85), to our knowledge, there has been no previous report of the in vitro production of a 3-dimensional cardiac ECM.
It is increasingly recognized that ECM is highly tissue-specific, with the fibroblasts of a given tissue synthesizing an ECM having a unique combination of structural proteins and bioactive molecules (i.e. growth factors). Accordingly, transplantation of cells across different tissue types could be problematic (see Badylak, S. F., D. O. Freytes, and T. W. Gilbert, Extracellular matrix as a biological scaffold material: Structure and function. Acta Biomater, 2009. 5(1): p. 1-13).
In addition to not being of myocardial origin, biomaterials currently under investigation for use in cardiac cell transfer patches have other notable limitations. For example, a frequent issue with the use of synthetic or decellularized tissues in patches for therapeutic cell delivery to the myocardium is the inability of the patch to physically adhere to the epicardial surface. The patches often require the use of glue or sutures to hold the patch to the heart (see, e.g., Fiumana, E., et al., Localization of mesenchymal stem cells grafted with a hyaluronan-based scaffold in the infarcted heart. J Surg Res, 2012). If the patch does not maintain firm contact with the surface of the heart, the ability of the cells to transfer is decreased significantly. Furthermore, epicardial patches must not only adhere to the heart, but must also have the proper tensile strength and compliance to tolerate cardiac movement. If a patch's compliance does not match that of the ventricle and does not move with the beating heart, gaps may form under the surface, reducing cell transfer. Epicardial patches lacking tensile strength may disintegrate under the strains of a beating heart.
For these reasons, a patch made from a 3-dimensional bioscaffold that is cardiac-specific would be highly desirable to facilitate successful delivery of therapeutic cells to injured or diseased myocardial tissue.